Expression of a protein in myocardium by injection of a gene

ABSTRACT

A novel method for expressing a protein which comprises: transforming skeletal myoblasts or cardiac myocytes with a DNA sequence comprising a DNA segment encoding a selected gene downstream of the Rous sarcoma virus long terminal repeat or the expression sequence in pRSV, and implanting said skeletal myoblasts or cardiac myocytes into a recipient which then expresses a physiologically effective level of said protein.

This application is a Continuation of application Ser. No. 07/789,983,filed on Nov. 12, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of skeletal and muscle cardiaccells that continuously secrete recombinant protein products.

2. Discussion of the Background

A large number of inherited and acquired diseases require treatment byintravenous or subcutaneous infusions of proteins. These includeinherited protein deficiencies such as hemophilia A and B, andcongenital growth hormone deficiency, as well as acquired diseases suchas AIDS and diabetes mellitus. Standard therapy for such diseasesrequires repeated intravenous or subcutaneous administration of proteinsolutions. The ability to use cell implants which continuously secreterecombinant protein products into the circulation would greatly simplifythe therapy of many of these disorders. This disclosure describestransplantable genetically-engineered skeletal muscle stem cells(myoblasts) that produce detectable levels of serum proteins in arecipient host and myocytes that produce local levels of secretedrecombinant proteins in the myocardium.

Somatic gene therapy can be defined as the ability to program theexpression of foreign genes in non-germ line (i.e., non-sperm and egg)cells of an animal or patient. Recent advances in molecular biologyincluding the cloning of many human genes and the development of viraland chemical gene delivery systems have brought us to the threshold ofsomatic gene therapy. All methods of gene therapy can be divided intotwo categories: ex vivo gene therapy involves the removal of cells froma host organism, introduction of a foreign gene into those cells in thelaboratory, and reimplantation or transplantation of the geneticallymodified cells back into a recipient host.

In contrast, in vivo gene therapy involves the introduction of a foreigngene directly into cells of a recipient host without the need for priorremoval of those cells from the organism. There are a number ofrequirements that must be met by any method of gene therapy before itcan be considered potentially useful for human therapeutics. First, onemust develop an efficient method for introducing the foreign gene intothe appropriate host cell. Secondly, it would be preferable to developsystems that program expression of the gene only in the appropriate hostcell type, thus preventing expression of the foreign gene in aninappropriate cell. Finally, and most importantly when considering humangene therapy, the technique must have a minimal risk of mutating thehost cells and of causing a persistent infection of the host organism, aparticularly important worry when using virus vectors to introduceforeign genes into host cells. This application pertains to novelsomatic gene therapy for use in heart and skeletal muscle which meetsthese requirements.

A. In Vivo Gene Therapy in Cardiac Myocytes

The ability to program recombinant gene expression in cardiac myocytesenables the treatment of a number of inherited and acquired cardiacdiseases. Therapeutic applications of this approach can be divided intoseveral general categories. First, to correct genetic disorders ofmyocardial cells. For example, injection of the normal dystrophin cDNAcan be used to correct the defects in cardiac contractility seen inpatients with Duchenne's muscular dystrophy. Secondly, to stimulate newcollateral circulation in areas of chronically ischemic myocardium byinjecting plasmids encoding recombinant angiogenesis factors directlyinto the left ventricular wall. Also, this approach can be used todirectly study the molecular mechanisms regulating cardiac myocyte geneexpression both during cardiac myogeneses and in a variety ofpathophysiologic states such as cardiac hypertrophy.

As many as 1.5 million patients per year in the U.S. suffer a myocardialinfarction (MI). Many millions more suffer from syndromes of chronicmyocardial ischemia due to large and small vessel coronaryatherosclerosis. Many of these patients will benefit from the ability tostimulate collateral vessel formation in areas of ischemic myocardium.The direct DNA injection method provides an alternative approach to thecurrent methods of coronary artery bypass and percutaneous transluminalcoronary angioplasty. In particular, many patients have such severe anddiffuse atherosclerosis that they are not candidates for CABG or PTCA.Thus far, there has been no approach which has successfully stimulatedcollateral vessel formation in areas of ischemic myocardium.

A number of genetic disorders affect myocardial performance. Forexample, many patients with Duchenne's muscular dystrophy also sufferfrom a cardiomyopathy. In addition, it is clear that there are a numberof other genetically-inherited cardiomyopathies of unknown etiology. Thegene injection approach described in this disclosure is useful fortreating a variety of these inherited disorders of cardiac function. Forexample, injection of vectors containing the normal dystrophin gene orcDNA can correct the defect in patients with Duchenne's musculardystrophy. Some aspects of the natural expression of the dystrophin genein muscle from DMD patients are discussed by Scott et al, Science, 239:1418 (1988). As additional genes for inherited cardiomyopathy areidentified, these gene products might also be injected into hearts inorder to correct abnormal cardiac function.

An understanding of the molecular mechanisms that regulatecardiac-specific gene expression during both normal cardiac developmentand a variety of pathological processes such as cardiac hypertrophy iscritical in designing rational therapeutic approaches to such problems.Previous approaches have all utilized in vitro transfection protocolsinto neonatal cardiocytes or transgenic approaches in mice. Such studiesare complicated by the fact that neonatal cardiocytes may not reflectthe in vivo situation and by the fact that neonatal cardiocytes have anextremely limited life span in tissue culture and cannot be incorporatedinto the heart. Moreover, transgenic approaches are lengthy (requiring 6months to 1 year) technically difficult and expensive. The geneinjection approach described in this disclosure obviates these problemsbecause it allows the stable expression of recombinant gene products inboth neonatal and adult cardiac myocytes in vivo in as little as 5 daysfollowing injection of DNA.

B. Ex Vivo Gene Therapy using Skeletal Myoblasts

A variety of acquired and inherited diseases are currently treated byrepeated intravenous or subcutaneous infusions of recombinant orpurified proteins. These include diabetes mellitus, treated withsubcutaneous or intravenous injections of insulin, hemophilia A, treatedwith intravenous infusions of factor VIII, and pituitary dwarfism,treated with subcutaneous injections of growth hormone. The developmentof cellular transplantation systems that can stably produce and deliversuch recombinant proteins into the systemic circulation would representan important advance in our ability to treat such diseases. The idealrecombinant protein delivery system would utilize a cell that can beeasily isolated from the recipient, grown and transduced withrecombinant genes in vitro, and conveniently reimplanted into the hostorganism. This cell would produce large amounts of secreted recombinantprotein, and following secretion, this protein would gain access to thecirculation. Finally, such implanted, genetically engineered cellsshould survive for long periods of time and continue to secrete thetransduced protein product without themselves interfering with thefunction of the tissue into which they were implanted.

Several different cellular systems have been used to produce recombinantproteins in vivo. These include karatinoxytes (M. Flowers et al, PNAS(USA) 87, 2349 (1990)), skin fibroblasts (T. D. Palmer, A. R. Thompson,A. D. Miller, Blood 73, 438 (1989); R. Scharfmann, J. H. Axelrod, I. M.Verma, PNAS (USA) 88, 4626 (1991)), hepatocytes (D. Armentary, A.Thompson, G. Darlington, S. Woo, PNAS (USA) 87, 6141 (1990); K. P.Ponder et al, PNAS (USA) 88, 1217 (1991)), lymphocytes (K. Culver et al,PNAS (USA) 88, 3155, (1991)), and bone marrow (E. Dzierzak, T.Papayannopoulou, R. Mulligan, PNAS (USA) 87, 439 (1990); M. Kaleko, J.V. Garcia, W. R. A. Osborne, A. D. Miller, Blood 75, 1733 (1990)).Although several of these systems have produced detectable levels ofcirculating proteins, it has proven difficult to produce stable,physiological levels of circulating recombinant proteins in normalanimals.

Burnetti et al, J. Biol. Chem, 265: 5960 (1990) have studied theregulation of myogenin and the events that occur when myoblaststransform into myocytes. However, no expression of a recombinant DNAsequence was disclosed. Paulson et al, J. Cell Biol., 110: 1705 (1990)describes the temperature-sensitive expression of all-torpedo andtorpedo-rat hybrid acetylcholine receiptor (AChR in mammalian musclecells. However, this expression of protein was performed in mousefibroblasts in culture, and not in vivo. Obtaining expression ofphysiological serum levels of a protein through a cellular implant isunrelated to and unsuggested by simply obtaining in vitro expression.

Pramanik et al, Eur. J. Biochem., 172: 355 (1988). Showed thattranslation of P-40 mRNA is repressed in non-proliferating myotubes byusing nuclease S1 mapping to quantify the steady-state levels of P-40mRNA in subcellular fractions of both myoblasts and myotubes. It wasshown that the result of the subcellular distribution of this mRNA inproliferating myoblasts following inhibition of DNA synthesis bycitazene or arabinoside have shown that translation of P-40 mRNAcontinued in the absence of DNA synthesis. This observation suggeststhat an additional signal is necessary to block the translation of P-40mRNA in myotubes.

Arnold et al (J. Biol. Chem. 257: 9872 (1982)) studied expression ofglyceraldehyde-3-phosphate dehydrogenase mRNA in developing chick heartcells in cultures. The gap dehydrogenase mRNA was present in 5 hour olddividing myoblasts. This method is limited to in vitro proteinexpression and does not address the issue of whether skeletal myoblastswill produce secreted recombinant proteins following differentiationinto myotubes.

SUMMARY OF THE INVENTION

Accordingly, one object of the invention is to provide a method forexpressing physiological levels of recombinant proteins so as to treatinherited or acquired diseases which comprises: implanting skeletalmyoblasts into a patient suffering from said condition, wherein saidmyoblasts are transformed with a DNA sequence comprising a DNA segmentencoding a selected gene downstream of the Rous sarcoma virus longterminal repeat or the expression sequence in pRSV to thereby obtain aphysiologically effective level of the gene product of said selectedgene in the blood.

Another object of the invention is to directly transduce cardiacmyocytes in vivo with a DNA sequence comprising a DNA segment encoding aselected gene downstream of the Rous sarcoma virus long terminal repeator the expression sequence in pRSV, capable of expressing the geneproduct of said selected gene in the myocardium of the host organism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-C) --Secretion of hGH over time by the G19 clone.

FIGS. 2(A-B)--Expression of hGH in muscle and serum of C2C12myoblast-injected mice.

FIGS. 3(A-E)--Photomicrographs of BAG-infected C2C12 cells and musclefrom C3H mice injected with BAG-infected C2C12 cells.

FIGS. 4(A-B)--Schematic method for introducing foreign genes into heartmuscle cells.

FIGS. 5(A-D)--Photomicrographs showing expression of β-galactosidase inheart muscle.

FIG. 6--Summary of cardiac injections with the β-gal expression vector.

FIGS. 7(A-B)--Capillary growth after injection of heart wall with afibroblast growth factor expression vector.

FIGS. 8A(1)-(2)--Schematic representation of pRSVCAT and pRSVgal.

FIG. 8B--Transcriptional activity of the RSV LTR in rat neonatalcardiocytes in vitro.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Skeletal Myoblasts as a Protein Delivery System

A novel plasmid vector, pRSV Growth Hormone (pRSVGH) was constructed bycloning a commercially available human growth hormone gene into a Roussarcoma virus pRSV expression vector constructed in my laboratory.Murine C2C12 myoblasts were stably co-transfected with two plasmids: (i)the pRSVGH plasmid containing the human growth hormone gene under thecontrol of the Rous sarcoma virus long terminal repeat (RSV LTR) and(ii) the pRSVneo plasmid which encodes resistance to the antibioticG418. C2C12 is a continuous cell line that has been shown todifferentiate into non-dividing, multinucleated myotubes in vitro. Thesemyotubes express the full complement of myofibrillar proteins anddisplay contractile activity. After exposure to G418 to select forstable transfectants, 2 out of 24 clones were shown to produce andsecrete relatively high levels of hGH in vitro (2.5 and 9 ng/hr/10⁶cells) and one of these, G19, was expanded for further studies.

In an initial series of experiments, the in vitro production andsecretion of hGH by G19 cells was quantitated using a sensitiveradioimmunoassay (RIA). This assay was linear over a range of hGHconcentrations between 0.05 and 50 ng/ml (FIG. 1A). Levels of hGH in theculture medium of G19 cells increased in a linear fashion between 2 and24 hours, with a mean rate of production of 12 ng/hour/10⁶ cells (FIG.1B). After secretion, there was almost no degradation of hGH asevidenced by the finding that the levels of hGH from culturesupernatants of G19 cells did not decrease significantly afterincubation for 24 hours on monolayers of non-transfected C2C12 cells(FIG. 1C). Following differentiation into myotubes in vitro, thehGH-transfected G19 cells continued to secrete hGH at a rate of 6ng/hr/10⁶ cells. Thus, non-dividing myotubes retain the ability toproduce secreted proteins. Finally, the RIA used in these experimentswere specific for hGH and did not cross-react with murine GH (FIG. 2B).

To determine whether G19 cells could produce circulating levels of hGHin vivo, a total of 6×10⁶ G19 cells were injected intramuscularly into 6separate sites in the lower limbs of normal 4-week-old syngeneic C3Hmice. Control mice received identical injections of F17 cells, aG418-resistant clone of C2C12 that does not produce hGH. To preventpotential cellular or antibody-mediated immune responses by the C3H miceto the human growth hormone produced by the G19 cells, all mice receiveddaily injections of cyclosporin A (5 mg/kg, IM). Mice were killed 5 daysor 3 weeks after injections, and muscle from the site of injection aswell as serum were assayed for hGH (FIGS. 2A-B). Muscle lysates frommice injected with the G19 hGH-producing cells contained 1.01±0.34 ng/ml(mean±S.D.) of human growth hormone at 5 days and 2.43±0.97 ng/ml at 3weeks as compared to control-injected mouse muscle which contained0.01±0.01 ng/ml (P<0.0001). The serum from G19-injected animalscontained 0.16±0.08 ng/ml and 0.28±0.08 ng/ml of hGH at 5 days and 3weeks following injection, respectively. These values were significantlydifferent from those of control mice (0.01±0.02 ng/ml) (P<0.0005) atboth time points. Thus, hGH expression appeared to be stable for atleast 3 weeks in these animals.

It was of interest to compare the levels of hGH in the myoblast-injectedmice to physiological levels of hGh in human serum. GH is secreted in apulsatile fashion in humans with normal physiological levels rangingbetween 0.1 and 25 ng/ml. Absolute levels of hGH also vary dependingupon the type of sample tested and the particular assay system used.Serum samples from normal human volunteers (n=7) contained 0.27±01 ng/mlof hGH as compared to 0.28 ng/ml, the mean serum level of hGH in theG19-injected mice. Thus, serum from the G19-injected animals containedphysiological levels of hGH 3 weeks after a single injection of 6×10⁶hGH-transfected myoblasts. Finally, animals were also injected with6×10⁶ G19 cells without concomitant immunosuppression with cyclosporinA. Serum from these animals (n=4) contained 1.0±0.25 ng/ml of hGH 3weeks following myoblast injection. Thus, immunosuppression does notappear to be necessary for the short-term production of recombinantproteins following myoblast injection.

An important question regarding the long-term feasibility of myoblastinjections concerns the fate of the myoblasts after IM injection. Toaddress this question, C3H mice were injected with C2C12 myoblasts thathad been previously infected in vitro with the β-galactosidaseexpressing BAG retrovirus and shown to express high levels ofintracellular β-galactosidase (FIG. 3A). β-Galactosidase expressing blueC2C12 cells were observed as clusters within areas of normal muscle(FIGS. 3C-E). Whereas the BAG-infected C2C12 myoblasts displayed amononuclear fibroblast-like appearance when grown in high levels ofserum in vitro (FIG. 3A), following injection many of these cells fusedinto multinucleated myotubes (FIGS. 3C-E) similar to those observedafter the differentiation of C2C12 cells by growth in low serum in vitro(FIG. 3B). In no case were tumors detected in the muscle or other organsof the C2C12 myoblast-injected animals at either 5 days or 3 weeks afterinjection. Moreover, lysates from the non-injected upper limbs, hearts,livers, kidneys, and lungs of the G19-injected animals were devoid ofhGH activity demonstrating that the injected cells remained localized tothe site of injection. However, because C2C12 is a continuous cell line,an accurate assessment of the malignant potential of the injected G19cells will require long-term follow-up of these animals. Finally, theβ-galactosidase expression seen in vivo was not due to the infection ofendogenous muscle with helper virus from the BAG-infected C2C12 cellsbecause no helper virus could be detected by co-cultivation assays usingthese cells and because retroviruses are unable to infect non-dividingcells such as myotubes. Therefore, these results show thatgenetically-modified C2C12 cells can become incorporated into theinjected muscle by differentiating into multinucleated myotubes in vivo.

Genetically engineered myoblasts are a useful delivery system forrecombinant proteins in vivo. These cells can produce large amounts ofsecreted recombinant proteins. They can be stably introduced into muscleby simple IM injection, and their secreted protein products gain accessto the circulation. The finding that the technique can be used toproduce detectable levels of hGH is especially encouraging given theshort half-life of hGH (less than 20 minutes) as compared with those ofother serum proteins.

Although all of the studies described above were performed with theimmortalized C2C12 cell line, identical techniques are directlyapplicable to primary human myoblasts. Previous reports have clearlydemonstrated that primary human myoblasts can be readily isolated,expanded in vitro, and reinjected into muscle. Like the C2C12 myoblasts,normal myoblasts can become incorporated into the injected muscle bydifferentiating into multinucleated myotubes in vivo. The unique findingof the present invention is that it is possible to program theexpression of a secreted recombinant protein in myoblasts in vitro andthat, following injection of these myoblasts into normal animals, theyfuse into myotubes and continue to produce the secreted recombinantprotein which gains access to the circulation. Thus, analogoustechniques to those described could easily be used to transfect primarymyoblasts which could then be introduced by intramuscular injection intonormal animals or humans.

This same system can express a wide variety of serum proteins such ashuman factor VIII and factor IX as well as insulin. A number of othercellular implant systems have been tried for the production of serumproteins. These include keratinocytes and hepatocytes. To our knowledge,none of these systems has successfully produced stable and physiologicallevels of a recombinant protein in normal animals.

B. In vivo Gene Therapy at Cardiac Myocytes Following direct Injectionof DNA

In another embodiment of the present invention it is possible to programrecombinant gene expression in cardiac myocytes after direct injectionof DNA into the left ventricular wall. Functional recombinant proteinexpression in myocytes was demonstrated directly using an enzymaticassay for β-galactosidase. Recombinant gene expression was observed inmyocytes from seven of nine of the injected hearts at both 3-5 days and3-4 weeks after injection. Expression was patchy and was observed onlyin direct contiguity with the site of injection. These findings haveseveral implications regarding both the use of this method for somaticgene therapy in the heart and the biology of recombinant DNA uptake andexpression in muscle cells.

The technique of somatic gene therapy using direct DNA injection intomyocardium has several advantages compared with other previouslydescribed methods of gene therapy. First, infectious viral vectors arenot required, eliminating the possibility of persistent infection of thehost. Second, a previous study (Wolff J A, Malone R W, Williams P, ChongW, Acsadi G, Jani A, Felgner P L: Direct gene transfer into mouse musclein vivo. Science 1990; 247: 1465-1468) has suggested that recombinantDNA taken up and expressed in skeletal myocytes persists as an episomeand therefore does not have the same potential for host cell mutagenesisas do retroviral vectors that integrate into the host chromosome.Finally, this method does not require the growth of recipient cells invitro, a requirement that would render transfection on nondividingcardiac myocytes particularly difficult.

Direct injection of recombinant DNA into the myocardium is useful forthe treatment of many acquired and inherited cardiovascular diseases inparticular, by stimulating collateral circulation in areas of chronicmyocardial ischemia by expressing recombinant angiogenesis factorslocally in the ventricular wall.

We have demonstrated that a recombinant bacterial β-galactosidase geneunder the control of the Rous sarcoma virus promoter can be introducedinto and expressed in adult rat cardiac myocytes in vivo by theinjection of purified plasmid DNA directly into the left ventricularwall. Cardiac myocytes expressing the recombinant β-galactosidaseactivity have been detected histochemically in rat hearts for at leastsix months following injection of the recombinant β-galactosidase gene.Rats have now been injected with the β-galactosidase gene for studies ofthe stability of expression at one year post-injection.

The method that we have used for introducing foreign genes into heartmuscle cells is shown schematically in FIG. 4. The method is mostremarkable for its simplicity. A concentrated solution of geneticmaterial (DNA) containing a single cloned gene is injected directly intothe beating heart wall in normal six-week-old rats. The particularcloned gene that was used in all of our initial studies is a bacterialgene called β-galactosidase. We chose this gene because it is notnormally expressed in heart cells. However, following injection of theDNA, we can detect its expression using a simple stain that turns cellsthat are expressing β-galactosidase blue. We were quite surprised tofind that following injection into a normal rat heart, theβ-galactosidase gene was taken up and expressed in heart muscle cells(FIGS. 5A-D). Thus, one can see areas of blue staining that correspondto the site of DNA injection and that represent upon high powermagnification, expression of the β-galactosidase gene in cells that areeasily identified as cardiac muscle by their striated pattern ofappearance. It should be emphasized that the only cells that we haveever observed to take up and express the DNA are heart muscle cells. Wehave never observed expression in other cells in the heart such asfibroblasts or the cells lining the heart blood vessels. As shown inFIG. 6, more than 75% of the hearts receiving the injected DNA expressthe foreign gene, and this expression is stable for periods of at leastsix months.

This method enables new blood vessel growth in areas of the heart thatare currently not receiving sufficient blood or oxygen. Several millionAmericans are currently afflicted with atherosclerotic narrowings of thecoronary arteries or blood vessels supplying the heart. In order todirectly stimulate new blood vessel growth in areas of the heart thatare not receiving sufficient blood because of coronary atherosclerosis,we have injected rat hearts with an anglogenesis factor gene, i.e. agene called fibroblast growth factor (FGF-5), that stimulates new bloodvessel growth. Following injection of FGF-5 DNA, we were able to show a30-40% increase in the number of capillaries in the injected heart wallas compared to hearts injected with control DNA solutions (FIG. 7).Microscopic examination revealed that the structure of the capillariesin the injected hearts was normal, thus suggesting that these newcapillaries can supply increased blood flow to the heart.

Rats have been injected with a plasmid-encoding human fibroblast growthfactor-5 (hFGF-5) in an attempt to stimulate anglogenesis or collateralblood flow in the adult rat heart. Rats were sacrificed at 3 weeksfollowing injection and capillary density was measured by computerizedlight microscopy. Rats injected with control vectors displayedapproximately 2300 capillaries/mm² at the site of injection. Incontrast, five animals injected with the FGF-5 expression vectordisplayed a 30-40% increase in capillary density with mean capillarydensities of approximately 3400/mm² (p<0.001). Thus, direct injection ofa fibroblast growth factor-5 expression vector stimulates collateralvessel formation in areas of injected myocardium.

In order to demonstrate recombinant gene expression in myocardium, anovel plasmid vector was constructed in which the bacterialβ-galactosidase gene from the PMSV β-gal vector was cloned immediatelydownstream of the Rous sarcoma virus long terminal repeat (LTR). Thisvector was shown to produce high-level gene expression in rat neonatalcardiac myocytes following transfection in tissue culture. In order tointroduce this plasmid into rat cardiac myocytes in vivo, 250 gSprague-Dawley rats were anesthetized with pentobarbital plus ketamine,incubated, and ventilated with a Harvard respirator. A left lateralthoracotomy was performed to expose the beating heart and 100 mg of PRSVβ-gal plasmid DNA in 100 ml of phosphate-buffered saline, plus 5%sucrose was injected into the apical portion of the beating leftventricle using a 30 gauge needle. Wounds were closed and animals wereallowed to recover for three days to four weeks. Animals were sacrificedand hearts were removed, fixed with glutaraldehyde and stained withX-gal for detection of the β-galactosidase protein. In additionalexperiments, a plasmid vector in which the human fibroblast growthfactor-5 cDNA is under the transcriptional control of the Rous sarcomavirus promoter has been constructed and injected into rat hearts using asimilar technique.

It should be noted that the pRSV LTR used in these experiments isderived from a chicken virus. Previous studies have demonstrated thatthis element is very active in skeletal and cardiac muscle from a largenumber of species including human, mouse, and rat in vitro. Therefore,the results obtained with this expression vector in vivo in rats andmice should be directly applicable to humans since this element istranscriptionally active in human cells.

To increase transduction frequencies, similar injections into ischemicmyocardium and injections with DC electrical countershock can be used.Additional vectors utilizing our recently-described cardiac Troponin Cpromoter/enhancer increase levels of recombinant gene expression.

Other features of the invention will become apparent in the course ofthe following examples which are given for illustration of the inventionand are not intended to be limiting.

EXAMPLES

Cell Culture and Transient Transfections

Neonatal rat cardiac myocytes were isolated from 1-2-day-oldSprague-Dawley rats (Charles River Laboratories, Wilmington, Mass.) bycollagenase digestion (Engelmann et al J. Mol Cell Cardiol. 1988; 20:169-77). This method results in the isolation of more than 90% cardiacmyocytes. Twenty-four hours after isolation, 1×10⁶ freshly isolatedmyocytes in a 60-mm collagen-coated dish (Collaborative Research Inc.,Waltham, Mass.) were transfected with 15 μg of cesium chloridegradient-purified chloramphenicol acetyl transferase (CAT) reporterplasmid DNA plus 5 μg of pMSVβgal reference plasmid DNA as follows: 20μg of plasmid DNA was resuspended in 1.5 ml of Opti-MEM (GIBCO, GrandIsland, N.Y.) and added to 1.5 ml of Opti-MEM containing 50 μl oflipofectin reagent (BRL, Gaithersburg, Md.). The resulting mixture wasadded to one 60-mm plate of cardiac myocytes. After 5 hours at 37° C. in5% CO₂, 3 ml of Medium 199 plus 5% fetal bovine serum (FCS) (GIBCO) wasadded to the cells, and the mixture was incubated at 37° C. for 48hours. Cell extracts were prepared and normalized for protein contentusing a commercially available kit (Biorad, Richmond, Calif.). CAT andβ-galactosidase assays were performed as previously described. ParmacekM S, Bengur A R, Vora A J, Leiden J M: J Biol Chem 1990 (in press).

Plasmids

The promoterless pSVOCAT plasmid (Gorman C M, Moffat L F, Howard B H:Recombinant genomes which express chloramphenicol acetyl-transferase inmammalian cells. Mol Cell Biol 1982; 77: 1432-1436) and the pRSVCAT(Gorman C, Padmanabhan R, Howard B H: High efficiency DNA-mediatedtransformation of primate cells. Science 1983; 221: 551-553) plasmid inwhich transcription of the bacterial CAT gene is under the control ofthe RSV promoter have been described previously. The pRSVβgal plasmidwas constructed by cloning the 4.0-kb β-galactosidase gene from pMSVβgal(Donoghue M, Ernst H, Wentworth B, Nadal-Ginard B, Rosenthal N: Amuscle-specific enhancer is located at the 3' end of the myosinlight-chain 1/3 gene locus. Genes Dev 1988: 2: 1779-1790) intoHindIII/BamHI-digested pRSVCAT (FIGS. 8A-8B).

Injection of Recombinant DNA In Vivo

Six- to 11-week-old 250-g Sprague-Dawley rats were housed and cared foraccording to National Institutes of Health guidelines in the ULAMfacility of the University of Michigan Medical Center. Rats wereanesthetized with 20 mg/kg pentobarbitol i.p. and 60 mg/kg ketaminei.m., intubated, and ventilated with a Harvard (Harvard Apparatus, SouthNatick, Mass.) respirator. A left lateral thoracotomy was performed toexpose the beating heart, and 100 μg of plasmid DNA in 100 μl ofphosphate-buffered saline (PBS) containing 5% sucrose (PBS/sucrose) wasinjected into the apical portion of the beating left ventricle using a30-g needle. Control animals were injected with 100 μl of PBS/sucrosealone. The animals were killed 3-5 or 21-30 days after injection bypentobarbitol euthanasia; hearts were removed via a median sternotomy,rinsed in ice-cold PBS, and processed for β-galactosidase activity.

Histochemical Analysis

Three-millimeter cross sections of the left ventricle were fixed for 5minutes at room temperature with 1.25% glutaraldehyde in PBS, washedthree times at room temperature in PBS, and stained for β-galactosidaseactivity with X-gal (Biorad) for 4-16 hours as described by Nabel et al.(Nabel E G, Plautz G, Boyce F M, Stanley J C, Nabel G J: Recombinantgene expression in vivo within endothelial cells of the arterial wall.Science 1989; 244: 1342-1344). The 3-mm sections were embedded withglycomethocyrlate, and 4-7 μm sections were cut and counterstained withhematoxylin and eosin as described previously. (Nabel E G, Plautz G,Boyce F M, Stanley J C, Nabel G J: Recombinant gene expression in vivowithin endothelial cells of the arterial wall. Science 1989; 244:1342-1344). Photomicroscopy was performed using Kodak Ektachrome 200film and Leitz Laborlux D and Wild M8 microscopes.

RSV LTR promotes High-Level Gene Expression in Rat Neonatal CardiocytesIn Vitro

To test the transcriptional activity of the RSV LTR in rodent cardiacmyocytes, the pRSVCAT vector (Gorman C, Padmanabhan R, Howard B H: Highefficiency DNA-mediated transformation of primate cells. Science 1983;221: 551-553) in which expression of the bacterial CAT gene is under thecontrol of the RSV LTR was transfected into primary neonatal rat cardiacmyocytes using lipofectin. Two days after transfection, the cultureswere harvested and assayed for CAT activity. All transfections alsocontained 5 μg of the pMSVβgal plasmid to correct for differences intransfection efficiencies. The RSV LTR was able to increasetranscription of the CAT gene 87-fold compared with the promoterlesspSVOCAT control plasmid. The pRSVCAT-transfected cardiac myocyteextracts produced 95% acetylation in a standard thin-layerchromatography assay. By comparison, identically prepared extracts of3T3 or HeLa cells transfected with this same vector produced 22% and 35%acetylation, respectively. Because the activities of cotransfectedpMSVβgal reference plasmids were almost identical in all threetransfections, these results demonstrated that the RSV LTR programshigh-level transcription in primary cardiac myocytes in vitro.

The ability to unambiguously identify the cell types that are expressingrecombinant gene products is an important requirement of all animalmodels of gene therapy. Because the bacterial β-galactosidase reportergene (but not the bacterial CAT gene) allows direct histologicalvisualization of recombinant gene expression, we constructed a pRSVβgalvector in which bacterial β-galactosidase gene expression is regulatedby the RSV LTR promoter for further studies of recombinant geneexpression in vivo (FIG. 8A).

Expression of β-Galactosidase Gene in Rat Cardiac Myocytes AfterInjection of pRSVβgal DNA Into the Left Ventricular Wall In Vivo

100 μg of pRSVβgal DNA was resuspended in 100 μl of PBS containing 5%sucrose (PBS/sucrose) and injected via a 30-g needle directly into thebeating left ventricular wall of 6-11-week-old Sprague-Dawley rathearts. Control rats received injections of 100 μl of PBS/sucrosewithout DNA. Rats were killed either 3-5 days or 3-4 weeks afterinjection, and hearts were fixed and stained for β-galactosidaseactivity. β-Galactosidase activity as manifested by dark-blue stainingwas readily apparent to the naked eye in sections of three of four ofthe pRSVβgal-injected hearts at 3-5 days and four of thepRSVβgal-injected hearts and 3-4 weeks after DNA injections. Thisstaining, which was focal and patchy, occurred only in a single area ofeach heart injected with pRSVβgal DNA and was not seen in five controlhearts injected with PBS/saline alone. Failure to observe staining intwo of nine of the pRSVβgal-injected hearts may have been due to thelack of DNA uptake or expression in these hearts or, more likely, totechnical difficulties in successfully centering and anchoring theneedle in the relatively thin beating left ventricular wall during theinjection process.

Because the normal ventricular wall contains both myocytes andfibroblasts and because the injection of DNA might be expected to causea localized inflammatory response, it was important to determine whichcell types were expressing the recombinant β-galactosidase gene.Histochemical analysis of sections from hearts injected with thepRSVβgal DNA clearly demonstrated β-galactosidase activity withincardiac myocytes that were easily identified by their myofibrillararchitecture. Between one and 10 positively staining myocytes were seenper high-power field, and these were often noncontiguous, suggestingthat the uptake of DNA and/or its expression is a relativelylow-frequency event. Because it was difficult to accurately identify theextent of DNA injection and because the positively staining area werequite focal and patchy, it was impossible to accurately quantitateeither the percentage or the total number of cells expressingrecombinant β-galactosidase activity in a given heart. However, it isclear that only a small fraction of cardiac myocytes expressed therecombinant protein. In addition, it is worth noting that sections fromthe 3-5 day postinjection hearts often showed evidence of an acuteinflammatory response along the track of the needle and that in severalcases fibrosis along the needle track was observed in sections from3-4-week postinjection hearts.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method for expressing a protein in cardiacmyocytes which comprises: injecting an expression vector containing aDNA segment encoding a selected gene into the myocardium of a mammalianhost, and obtaining expression in cardiac myocytes of the proteinencoded by said selected gene.
 2. The method of claim 1, wherein saidexpression vector is injected into the ventricular wall.
 3. The methodof claim 1 wherein said expression vector contains a DNA segmentencoding a selected gene downstream of the Rous sarcoma virus longterminal repeat or the expression sequence in pRSV.